The present invention generally relates to the use of the arrestin-2/STAM-1 complex as a therapeutic target, for example, to identify and develop pharmacological agents capable of treating medical diseases, such as the treatment of metastasis in cancer patients and myocardial infarction.
Chemokines are a family of small cytokines, or proteins, that are secreted by cells of certain organisms, and in particular the cells of all vertebrates. Chemokines interact with G protein-linked transmembrane receptors, or chemokine receptors, found on the surfaces of their target cells. Of interest to the present invention are the CXC family of chemokines (α-chemokines), and in particular the CXC chemokine receptors (CXCR) to which CXC chemokines bind.
The CXC chemokine receptor 4 (CXCR4), a G protein-coupled receptor (GPCR), upon activation by its cognate ligand stromal-cell derived factor-1α (SDF-1α/CXCL12), is known to be rapidly internalized and targeted into the degradative pathway by a ubiquitin-dependent mechanism. See Marchese, A., and Benovic, J. L., Agonist-promoted ubiquitination of the G protein-coupled receptor CXCR4 mediates lysosomal sorting, J. Biol. Chem. 276, 45509-45512 (2001); Shenoy, S. K., McDonald, P. H., Kohout, T. A., and Lefkowitz, R J., Regulation of receptor fate by ubiquitination of activated beta 2-adrenergic receptor and beta-arrestin, Science 294, 1307-1313 (2001); and Marchese, A., Raiborg, C., Santini, F., Keen, J. H., Stenmark, H., and Benovic, J. L., The E3 ubiquitin ligase AIP4 mediates ubiquitination and sorting of the G protein-coupled receptor CXCR4, Dev. Cell 5, 709-722 (2003). Activation by CXCL12 induces rapid and transient phosphorylation of serine residues 324 and 325 within the carboxyl-terminal tail (C-tail) of CXCR4, thereby promoting binding to the E3 ubiquitin ligase atrophin-I interacting protein 4 (AIP4) via a novel WW-domain mediated interaction culminating in ubiquitination of vicinal lysine residues (Marchese et al. (2003); Bhandari, D., Robia, S. L., and Marchese, A., The E3 ubiquitin ligase atrophin interacting protein 4 binds directly to the chemokine receptor CXCR4 via a novel WW domain-mediated interaction, Mol. Biol. Cell. 20, 1324-1339 (2009)). This is followed by internalization of CXCR4 onto early endosomes where the ubiquitin moiety serves as a sorting signal to direct the receptor to lysosomes for proteolysis (Marchese and Benovic (2001); Marchese et al. (2003)).
In general, the ubiquitin moiety on ubiquitinated receptors interacts with ubiquitin binding domains (UBD) found in several proteins of the endosomal sorting complex required for transport (ESCRT) machinery (Raiborg, C., and Stenmark, H., The ESCRT machinery in endosomal sorting of ubiquitylated membrane proteins, Nature 458, 445-452 (2009); Shields, S. B., Oestreich, A. J., Winistorfer, S., Nguyen, D., Payne, J. A., Katzmann, D. J., and Piper, R., ESCRT ubiquitin-binding domains function cooperatively during MVB cargo sorting, J. Cell Biol. 185, 213-224 (2009)). The ESCRT machinery is made up of four distinct protein complexes (ESCRT 0-III) that act in a sequential and coordinated manner to target ubiquitinated receptors into multivesicular bodies, which then fuse with lysosomes where degradation occurs. Recruitment into this pathway takes place by the initial recognition of the ubiquitinated receptor by ESCRT-0, which then subsequently recruits ESCRT-I to the endosomal membrane, followed by recruitment of ESCRT II and III, culminating in proper execution of the sorting process (Williams, R. L., and Urbe, S., The emerging shape of the ESCRT machinery, Nat. Rev. Mol. Cell Biol. 8, 355-368 (2007); Raiborg and Stenmark (2009)). Hepatocyte growth factor-regulated tyrosine kinase substrate (HRS) is understood to be a critical element of ESCRT-0 and has been shown to mediate down regulation of several cell surface signaling receptors (Bache, K. G., Brech, A., Mehlum, A., and Stenmark, H., Hrs regulates multivesicular body formation via ESCRT recruitment to endosames, J. Cell Biol. 162, 435-442 (2003); Kanazawa, C., Morita, E., Yamada, M., Ishii, N., Miura, S., Asao, H., Yoshimori, T., and Sugamura, K., Effects of deficiencies of STAMs and Hrs, mammalian class E Vps proteins, on receptor downregulation, Biochem. Biophys. Res. Commun. 309, 848-856 (2003); Abella, J. V., Peschard, P., Naujokas, M. A., Lin, T., Saucier, C., Urbe, S., and Park, M., Met/Hepatocyte growth factor receptor ubiquitination suppresses transformation and is required for Hrs phosphorylation, Mol. Cell Biol. 25, 9632-9645 (2005); Hasdemir, B., Bunnett, N. W., and Cottrell, G. S., Hepatocyte growth factor-regulated tyrosine kinase substrate (HRS) mediates post-endocytic trafficking of protease-activated receptor 2 and calcitonin receptor-like receptor, J. Biol. Chem. 282, 29646-29657 (2007)). One such cell surface signaling receptor is CXCR4 (Marchese et al. (2003)). The ubiquitin moiety on CXCR4 is thought to interact with the ubiquitin interacting motif (UIM) found in HRS, thereby targeting CXCR4 into the degradative pathway.
Together with HRS, signal-transducing adaptor molecule (STAM) forms ESCRT-O, STAM was originally identified as an adaptor protein involved in cytokine signaling (Takeshita, T., Arita, T., Asao, H., Tanaka, N., Higuchi, M., Kuroda, H., Kanecko, K., Munakata, H., Endo, Y., Fujita, T., and Sugamura, K.; Cloning of a novel signal-transducing adaptor molecule containing an SH3 domain and ITAM, Biochem, Biophys, Res. Commun. 225, 1035-1039 (1996); Takeshita, T., Arita, T., Higuchi, M., Asao, H., Endo, K., Kuroda, H., Tanaka, N., Murata, K., Ishii, N., and Sugamura, K.; STAM, signal transducing adaptor molecule, is associated with Janus kinases and involved in signaling for cell growth and c-myc induction, Immunity 6, 449-457; (1997). Two STAM isoforms exist, STAM-1 and STAM-2, which share 53% amino acid identity and may be redundant in their function (Lohi, O., Poussu, A., Merilainen, J., Kellokumpu, S., Wasenius, V. M., and Lehto, V. P., EAST, an ipidermal growth factor receptor- and Eps 15-associated protein with Src homology 3 and tyrosine-based activation motif domains, J. Biol. Chem., 273, 21408-21415 (1998); Endo, K., Takeshita, T., Kasai, H., Sasaki, Y., Tanaka, N., Asao, H., Kikuchi, K., Yamada, M., Chenb, M., O'Shea, J. J., and Sugamura, K., STAM2, a new member of the STAM family, bindign to the Janus kinases, FEBS Lett, 477, 55-61 (2000); Pandey, A., Fernandez, M. M., Steen, H., Blagoev, B., Nielsen, M. M., Roche, S., Mann, M., and Lodish, H. F., Identification of a novel immunoreceptor tyrosine-based activation motif-containing molecule, STAM2, by mass spectrometry and its involvement in growth factor and cytokine receptor signaling pathways, J. Biol. Chem., 275, 38633-38639 (2000); Yamada, M., Ishii, N., Asao, H., Murata, K., Kanazawa, C., Sasaki, H., and Sugamura, K., Signal-transducing adaptor molecules STAM1 and STAM2 are required for T-cell development and survival, Mol. Cell Biol., 22, 8648-8658 (2002). Similar to HRS, STAM also binds to ubiquitin and may act in concert with HRS to recruit ubiquitinated receptors for lysosomal sorting (Asao, H., Sasaki, Y., Arita, T., Tanaka, N., Endo, K., Kasai, H., Takeshita, T., Endo, Y., Fujita, T., and Sugamura, K., Hrs is associated with STAM, a signal-transducing adaptor molecule, Its suppressive effect on cytokine-induced cell growth, J. Biol. Chem., 272, 32785-32791 (1997); Takata, H., Katao, M., Denda, K., and Kitamura, N., A hrs binding protein having a Src homology 3 domain is involved in intracellular degradation of growth factors and their receptors, Genes Cells 5, 57-69 (2000); Bache, K. G., Raiborg, C., Mehlum, A., and Stenmark, H., STAM and Hrs are subunits of a multivalent ubiquitin-binding complex on early endosomes, J. Biol. Chem., 278, 12513-12521 (2003b); Kanazawa et al., (2003). STAMs may also modulate endosomal sorting by virtue of their ability to interact with endosomal associated deubiquitinating enzymes AMSH (associated molecule with the SH3 domain of STAM) and UBPY, which may modulate the ubiquitination status of both receptors and/or the sorting machinery (McCullough, J., Clague, M. J., and Urbe, S., AMSH is an endosome-associated ubiquitin isopeptidase, J. Cell Biol., 166, 487-492 (2004); Bowers, K., Piper, S. C., Edeling, M. A., Gray, S. R., Owen, D. J., Lehner, P. J., and Luzio, J. P., Degradation of endocytosed epidermal growth factor and virally ubiquitinated major histocompatibility complex class I is independent of mammalian ESCRTII, J. Biol. Chem., 281, 5094-5105 (2006); McCullough, J., Row, P.e., Lorenzo, O., Doherty, M., Beynon, R., Clague, M. J., and Urbe, S., Activation of the endosome-associated ubiquitin isopeptidase AMSH by STAM, a component of the multivesicular body-sorting machinery, Curr. Biol., 16, 160-165 (2006); Row, P. E., Prior, L. A., McCullough, J., Clague, M. J., and Urbe, S., The ubiquitin isopeptidase UBPY regulates endosomal ubiquitin dynamics and is essential for receptor down-regulation, J. Biol. Chem., 281, 12618-12624 (2006); Kong, C., Su, X., Chen, P. I., and Stahl, P. D., Rin1 interacts with signal-transducing adaptor molecule (STAM) and mediates epidermal growth factor receptor trafficking and degradation, J. Biol. Chem., 282, 15294-15301 (2007); Ma, Y. M., Boucrot, E., Villen, J., Affar el, B., Gygi, S. P., Gottlinger, H. G., and Kirchhausen, T., Targeting of AMSH to endosomes is required for epidermal growth factor receptor degradation, J. Biol. Chem., 282, 9805-9812 (2007). Recently, STAMs have been implicated in endoplasmic reticulum to Golgi trafficking, possibly via their interaction with coat protein II proteins (Rismanchi, N., Puertollano, R., and Blackstone, C., STAM adaptor proteins interact with COPII complexes and function in ER-to-Golgi trafficking, Traffic 10, 201-217 (2009). However, their role in GPCR trafficking and signaling is believed to be relatively unknown.
It has been recently shown that arrestin-2 mediates endosomal sorting of CXCR4 (Bhandari, D., Trejo, J., Benovic, J. L., and Marchese, A., Arrestin-2 interacts with the ubiquitin-protein isopeptide ligase atrophin-interacting protein 4 and mediates endosomal sorting of the chemokine receptor CXCR4, J. Biol. Chem., 282, 36971-36979 (2007). Non-visual arrestins, arrestin-2 and arrestin-3 (also known as β-arrestin1 and β-arrestin2, respectively), are generally known for their ability to regulate GPCR desensitization, internalization and signaling (Moore, C. A., Milano, S. K., and Benovic, J. L., Regulation of receptor trafficking by GRKs and arrestins, Ann. Rev. Phy., 69, 451-482 (2007), although their role in endosomal sorting remains relatively unexplored. Arrestin-2 interacts with and co-localizes with AIP4 on early endosomes, where it targets CXCR4 for lysosomal sorting (Bhandari et al., 2007). In addition to mediating ubiquitination of CXCR4 at the plasma membrane, AIP4 also interacts with and mediates ubiquitination of HRS, likely on endosomes. However, the function of the ubiquitin moiety remains unknown (Marchese et al., 2003). How arrestin-2 may integrate with AIP4 and HRS to carry out CXCR4 sorting into the degradative pathway remains poorly understood.
It is believed that others have used pharmacological agents that directly target CXCR4 to antagonize agonist (CXCL12) evoked CXCR4 signaling mediated events. A major disadvantage of this approach is that directly targeting CXCR4 is not specific, as it would modulate all intracellular signaling cascades activated by CXCR4. A major caveat with this approach is that it has the potential of producing unintended consequences, such as deleterious side-effects.